Secondary Metabolites and Biological Activities of Talaromyces sp. LGT-2, an Endophytic Fungus from Tripterygium Wilfordii.

In the present study, eleven compounds (1-11) including nine alkaloids (1-9), one triterpenoid saponin (10) and one formamide (11) were isolated from Talaromyces sp. LGT-2, an endophytic fungus from Tripterygium wilfordi. Their structures were determined based on NMR and ESI-MS spectral data, as well as comparing previous literature data. This is the first report of the isolation of alkaloids (1-9) from Talaromyces genus. In the next step, all compounds were screened for their anti-monoamine oxidase, anti-acetylcholinesterase, antibacterial and antitumor activities. Compound 11 showed moderate anti-monoamine oxidase activity with IC50 value of 61 μM; compounds 3, 4, 8 showed weaker anti-acetylcholinesterase activity; compounds 1, 3, 4, 7, 8, 9 showed moderate antibacterial activities; compounds 7, 8, 9 showed cytotoxicity against B16 cancer cell line with inhibitory rate of 86%, 82%, 78%, respectively, at the concentration of 500 μg/mL.


Introduction
Endophytic fungi have been proved to be a new source for natural compounds, literature reports that we can get secondary metabolites which have unique structure and wide range of biological activities, such as antitumor, antimicrobial and antituberculosis. Indeed , structural diversity of these metabolites make endophytic fungi a potential new lead for drug discovery and development (1,2).
During our ongoing screening for new bioactive natural products from endophytes, we found the fermentation broth of Talaromyces sp.
LGT-2 (GenBank Accession No. KF934203), an endophytic fungus inhabited in Tripterygium wilfordi, showed moderate monoamine oxidase (MAO) inhibitory activity with IC 50 value of 85 μg/mL. Further chemical investigation resulted in the isolation of compounds 1-11 (Figure 1.). Anti-MAO activity, anti-acetylcholinesterase (anti-AChE), antitumor and antibacterial activities of compounds 1-11 were also evaluated in this study (Table 1.). LGT-2 was isolated from Chinese herb medicine Tripterygium wilfordii and was identified as Talaromyces sp. based on both morphology on PDA and analysis of the DNA sequences of the ITS1-5.8S-ITS2 ribosomal DNA gene region (GenBank Accession No. KF934203).
Antimicrobial assay: The antimicrobial assay was performed by measuring zones of inhibition (mm) using standard disc diffusion technique (3). A positive control, amoxicillin (0.1mg/ mL) was used for comparison purpose, whilst a blank disc impregnated with appropriate solvent was used as a negative control. In addition, the minimum inhibitory concentrations (MIC) of all the monomer compounds against Escherichia coli, Pseudomonas Aeruginosa, Staphylococcus aureus, Bnfillus licheniformis, Streptococcus pneumoniae, were determined by serial dilution technique.
Cytotoxicity Bioassay: The cytotoxicity of compounds 1-11 against B16 cancer cell line was measured by the MTT method(4). Cyclophosphamide was used as positive control.
Anti-MAO and anti-AChE Bioassay: The procedure of testing MAO and AChE inhibiting activity was same with that reported in our previous paper (5, 6).

Results and Discussion
This study was focused on compounds isolated from second metabolites of Talaromyces sp.